Cloning And Functional Analysis Of The Duplicated DGAT1Genes In Brassica Napus

Oilseed rape is one of the four main oil crops in the world. The cultivated area ofthe oilseed rape is less than soybean as well as cotton. The Brassica napus is the maincultivated species of the oilseed rape in China, increasing its oil content can release theameliorate our tight market.In plant, triacylglycerol （TAG） is the most important lipid storage form.Diacylglycerol acyltransferases （DGAT） is the key enzyme of the TAG biosyntheticpathway and catalyzes fatty acyl group to the sn-3of diacylglycerol （DAG）.Acyl-CoA:diacylglycerol acyltransferase1（DGAT1） is the key enzyme that has beenconfirmed to contribute to TAG biosynthesis in Arabidopsis thaliana seeds. PreviouslyResearches showed dgat1null mutants displayed only a20~40%decrease in the seed oilcontent, a consistently lower ratio of TAG/DAG, altered fatty acid composition, reducedgermination rates and reduced seed weight. In this research, we focus on the function ofBrassica napus DGAT1to improve B.napus seed oil content. There is only one copy ofDGAT1in Arabidopsis thaliana. But Brassica napus， as an allotetraploid plant, there may bemany copies in the genome of Brassica napus, which makes the cloning and functionalanalysis of DGAT1gene more difficult.The experimental material used in this study is Brassica napus. Based on thepublished DGAT1gene sequence of TAIR, sequence alignments was made in the BRADdatabase with the BLAST tool. Then we design primers on the basis of the alignmentresults--two DGAT1gene sequences from Brassia rapa and the other two from Brassicaoleracea. RT-PCR was utilized to obtain four DGAT1gene fragments from Brassica napus,which were sequenced and bioinformatics analysis was carried out afterwards. And then theexpression of these BnDGAT1-a and BnDGAT1-b genes were analyzed in developingB.napus seeds with different oil contents. Plant expression vectors were constructed forexpressing those four DGAT1genes in B.napus with Gateway technology. Columbiawild-type and dgat1null mutant Arabidopsis thaliana were transformed by using flowerdipping method and oil contents of those lines were measured with gas chromatography toidentify the function of those duplicated DGAT1genes. The results of detailed research were as follows:1） Four DGAT1gene fragments including coding region from Brassica napus wereobtained by RT-PCR, which were sequenced and bioinformatics analysis was carried out.Results suggested that the lengths of four cloned gene fragments were1509bp（BnDGAT1-1）,1533bp （BnDGAT1-2）,1515bp （BnDGAT1-3） and1506bp （BnDGAT1-4） respectively.These four genes had very high similarity, which was up to97％between BnDGAT1-1andBnDGAT1-2. The same similarity appeared between BnDGAT1-3and BnDGAT1-4. Theanalysis of transmembrane structure showed that protein coded by either of the pair ofBnDGAT1-1and BnDGAT1-2genes contains9transmembrane domains, while8transmembrane domains were found in BnDGAT1-3and BnDGAT1-4coding protein. Theexpression analysis of these BnDGAT1-a and BnDGAT1-b genes in developing seeds withdifferent oil contents showed no significant differences, which suggested that expression ofthese two pairs of DGAT1was not the critical factor determining oil contents of the testedseeds.2） In this research, we transformed four duplicated DGAT1genes fromBrassica.napus which had been obtained by Gateway techniques into Arabidopsis wild typeCol-0and dgat1knock-out mutant AS11. The gas chromatography analysis of seeds from T2generation transformant showed that the oil content of both Col-0and AS11have dramaticallyraised, which indicated that all these four duplicated genes were functional. According to thedata analysis, the overexpression of BnDGAT1-1、BnDGAT1-2、BnDGAT1-3、BnDGAT1-4caused a38.18%、55.05%、42.55%、47.22%raise of seeds oil content in average in Col-0transformant in compared with original plant. The analysis also showed that theoverexpression of BnDGAT1-1、BnDGAT1-2、BnDGAT1-3、BnDGAT1-4caused a8.11%、44.56%、16.34%、30.86%raise in AS11transformant seeds oil content in compared withoriginal plant. In transformant of both Col-0and AS11plant, we detected that the content ofC18:2、C18:3、C20:1fatty acids were higher in compared with original plant, while theC16:0、C18:0fatty acid were lower than before.